GFR alpha2/neurturin signalling regulates noxious heat transduction in isolectin B4-binding mouse sensory neurons

Abstract

The GFR alpha2 receptor is the cognate co-receptor for the neurotrophic factor neurturin and GFR alpha2 is selectively expressed by isolectin B(4) (IB(4))-binding nociceptive sensory neurons. Here, we used two physiological approaches in combination with mice that have a targeted deletion of the GFR alpha2 gene (GFR alpha2 -/- mice) in order to determine whether GFR alpha2/neurturin signalling regulates the functional properties or the survival of IB(4)-binding nociceptors. Because 50 % of IB(4)-binding neurons respond to noxious heat and because patch clamp recordings of isolated dorsal root ganglion sensory neurons allow one to neurochemically identify subpopulations of neurons, we analysed the noxious heat responsiveness of IB(4)-positive and -negative small-diameter neurons isolated from adult GFR alpha2 -/- and littermate control mice. The percentage of IB(4)-positive neurons that had large (> 100 pA) heat-evoked inward currents was severely reduced in GFR alpha2 -/- mice (12 %) compared to wild-type littermates (47 %), and this loss in large-magnitude heat currents was accounted for by an increase in neurons with very small (< 100 pA) heat-evoked currents as well as an increase in neurons with no detectable heat current. Counts of IB(4)-positive and -negative neurons, as well as counts of unmyelinated axons in the saphenous nerve, confirmed that the loss in neurons with large-amplitude heat currents was due to a deficit in heat transduction and not a decrease in cell survival. The effect was modality specific for heat because mechanical transduction of all fibre types, including IB(4)-positive C fibres, was normal. Our data are the first to indicate a transduction-function role for GFR alpha2/neurturin signalling in a specific class of sensory neurons.

Figure 1. IB₄-positive neurons with large heat currents are markedly reduced in GFRα2 −/− mice

A, examples of noxious heat-induced inward currents in an IB₄-positive and an IB₄-negative neuron from a wild-type mouse. B, the percentage of IB₄-positive neurons with heat-evoked currents > 100 pA was substantially reduced in GFR α2 −/− mice compared to wild-type mice (** P = 0.008, Fisher's exact test) whereas the percentage of heat-sensitive IB₄-negative neurons was unaltered. C, the loss in IB₄-positive neurons with large-magnitude (> 100 pA) heat currents was due to a shift toward neurons with small heat currents (< 100 pA) and to more neurons with no detectable heat current (0 pA).

Figure 2. GFR α2 −/− mice have smaller-diameter unmyelinated and myelinated axons in cutaneous nerves

A and B, cross-sections of the saphenous nerve at mid-thigh level from a wild-type (A) and a GFR α2 −/− (B) mouse. Calibration bar, 50 μm. Note that although no axons were lost (see Results), the nerves from GFR α2 −/− mice were considerably smaller than those from wild-type littermates. C-D, histograms show the distribution of axon diameters of unmyelinated (C) and myelinated (D) axons in the saphenous nerves from GFR α2 −/− (n = 4) and wild-type (n = 4) mice. Note the leftward shift in both classes of axons from GFR α2 −/− mutants.

Figure 3. GFR α2 −/− mice have normal numbers of IB₄-positive DRG neurons

A-B, histograms illustrate the distribution of cell bodies of IB₄-positive and -negative neurons in DRG cultures taken from GFR α2 +/+ (n = 3) and wild-type (n = 3) mice. There was no difference in the percentage of neurons that were IB₄ positive in the GFR α2 −/− mutants (48.8 %; 125/256) compared to the wild-type controls (48.8 %; 190/389).